This proposal represents the continuation of a project that hs been a part of our SCOR grant since its inception in 1987 and which has resulted in 21 peer-reviewed papers to date. In the first funding period, techniques were developed to allow assessment of osteoclast activity in animal models (rat and chick). This approach was used to examine the regulation of osteoclast activity by PTH, CT, and pH. In the second funding period, with the recognition that the amount of bone resorbed depends not only on the activity of individual osteoclasts, but also on their rate of recruitment, techniques were established to assess osteoclast formation, as well as activity, in murine marrow cultures. This approach was used to study the interactions of PTH and estrogen. While these animal models have yielded much useful data, the studies have indicated that there are marked species differences in osteoclast activity and, more importantly, in their regulation by calciotropic factors. This emphasizes the importance of using human cells if were are interested in extrapolating the results to normal and abnormal human skeletal biology. Until recently, however, the study of human osteoclasts has been hampered by difficulties in obtaining sufficient numbers of osteoclasts in culture that express the key phenotypical feature of osteoclasts, namely bone resorption. Quite recently, this problem has been overcome by the addition of M-CSF to cultures of osteoclast precursor cells derived from the peripheral circulation and bone marrow. In this present project we propose to capitalize on these new in vitro models of human osteoclast formation, in addition to "knockout" murine models, to increase our understanding of the mechanisms regulating the differentiation and death of osteoclasts. The project is divided into three specific aims: 1. To investigate the regulation of human osteoclast differentiation and isolate a purified population of pre-fusion osteoclasts. 2. To investigate the role of estrogen receptors alpha and beta in regulating murine and human osteoclast formation. 3. To investigate the role of apoptosis in mediating estrogen's effects on human and murine osteoclast formation and life span. Although designed as an independent project, this cell biology project is well integrated with the other projects of the SCOR as the effects of the two agents of central interest (PTH and estrogen) will be studied in osteoclasts in vitro. There are numerous areas in which this project integrates with the other projects. For example, this project studies the regulation of osteopontin synthesis in osteoclasts and in Project 2 this will be studied in osteoblasts. Moreover, questions that will be pursued in the animal and clinical studies (e.g., does intermittent administration of PTH have different effects on osteoclast recruitment than continuous administration?) will also be tackled in this project.